Project 1 serves the program by testing the hypothesis that acute acidification by lactic acidosis will enhance thermosensitization and selectively sensitize melanoma xenografts to radiotherapy. Protocols were established for acute acidification to less than or equal too pH3 6.3 and oxygenation of melanoma has uniquely high activity of H+ linked monocarboxylate transporters (MCT, a.k.a. lactate symport) that are the major membrane proton exchange. Although thermosensitization by acidification was demonstrated, radiation must be combined with hyperthermia to achieve local control. Five specific aims will test the hypothesis and investigate mechanisms of melanoma acidification. Aim 1 will investigate inhibition of mitochondrial respiration by meta-iobenzylguanidine (MIBG) on melanoma acidification and oxygenation for sensitization to thermoradiotherapy. Variation in glycolytic/respiratory ratio among frozen melanoma biopsy cell suspensions will indicate relative susceptibility to MIBG. Aim 2 will investigate inhibition of the MCT either by alpha-cyano-4-hydroxy-cinnamic acid (CNCn) or by Ionidamine on acidification of melanoma and sensitization to thermoradiotherapy. The activity of the plasma membrane MCT among stored melanoma biopsy cell suspensions will indicate relative susceptibility to CNCn or Ionidamine. Aim 3 will identify the utility of combining MIBG and CNCn as a cocktail to acidify and oxygenate all melanomas. Melanomas with high rates of respiration will be sensitive to MIBG, and those with high rates of glycolysis sensitive to CNCn. In each protocol excess glucose must be combined with inhibitors to fuel lactate production. Two different F2 melanomas xenografts in nude mice will be studied that differ in vascularity, radiation sensitivity and expression of heat shock proteins. Tumor growth delay will be compared with tibial bone marrow toxicity to establish therapeutic gain. Cellular radiation response parameters in vitro will be determined in Project 3. Project 1 provides mice with melanoma xenografts to Project 2, so that together with Core A, biological endpoints can be investigated to predict response of melanomas and normal tissues to treatment pH9, pHi, 23Nai, pO2, oxygen consumption, bioenergetics and blood flow. Aim 4 supports Project 3 to determine the effect of acute acidification on apoptosis and the relationship with hyperthermia-induced upregulation of hsp70 in DB-1 xenografts grown up from cell stably transfected with the gene for enhanced green fluorescent protein (egfp) under control of the hsp70 promoter. Aim 5 with Project 3 supports Project 4 to confirm whether nucleolin translocation is a determinant of heat response in vivo by measuring nucleolin movement from the nucleolus into the nucleo-plasm in DB-1 xenografts as a function of acidification during hyperthermia. Acute tumor acidification will have the clinical effect of increased tumor temperature with additional benefit from increased tumor oxygenation. These results will help direct a Phase I/II trial conducted by Dr. Douglas Fraker, Univ. of Penn. To demonstrate the effects of acidification of melanoma response to limb perfusion with melphalan at 42 degrees Centigrade.